PROJECT SUMMARY/ABSTRACT Obesity affects approximately 35% of adults and 17% of children in the United States increasing the risks of heart disease, stroke and type 2 diabetes. The human gut microbiota, the trillions of microbes that inhabit the gastrointestinal tract, have been implicated as an environmental factor linked to obesity and energy balance; however, the mechanisms are not fully understood. Diet remains the first line intervention to induce weight loss, but its impact on the microbiota, and how this may affect weight loss and regain remain unclear. My preliminary results from a very-low calorie diet intervention in human subjects reveal that caloric restriction induces antibiotic-like disturbances in microbiota composition and function. Fecal transplant from post-diet humans to germ-free mice induces weight loss. Analysis of both human and mouse microbiotas revealed that the diet-induced reconfiguration of the microbiota allowed for expansion of Clostridioides [Clostridium] difficile, best known as a major cause of antibiotic-associated diarrhea and its severe complications. In a colonization model, C. difficile was sufficient to drive weight loss, reduce body fat, and increase glucose tolerance without causing acute disease. These observations have led to the hypothesis that diet interactions with the gut microbiota and C. difficile disrupt nutrient uptake contributing to energy imbalance. The first aim of these studies will focus on the ability of C. difficile to affect host energy balance while characterizing the mechanisms through which it occurs. Preliminary data strongly implicates the C. difficile toxins TcdA and/or TcdB. Using combinatorial and individual knockouts, the causative toxin will be identified and its effects on host energy balance will be extensively characterized. To define the mechanisms through which C. difficile acts at the level of the intestinal epithelium, the effect of sub-toxic purified toxin(s) on nutrient absorption and cell physiology will be examined in organoid models of both the human and mouse intestine. Finally, the ability of asymptomatic colonization to counter diet-induced obesity will be examined. The second aim of this work will examine the mechanism through which caloric restriction affects C. difficile permissibility. Specifically, this aim will test the hypothesis that caloric restriction depletes microbes that produce C. difficile-inhibitory secondary bile acids. Through a humanized mouse model of caloric restriction, and sequence-guided isolation and metabolic characterization, synthetic communities will be designed replicating diet-responsive microbes to specifically test the role of secondary bile acid biosynthesis, and potentially identify new antagonistic interactions which are of great relevance to C. difficile treatment and prevention. The proposed experiments in these aims will leverage my expertise in the microbiome field with new training in obesity and metabolic disease research. An expert interdisciplinary advisory committee, and an institutional focus on microbiome and metabolism research, will provide the ideal environment for the proposed scientific and professional development leading to the creation of an independent research program.